Defects in the equal partitioning of chromosomes at cell division causes aneuploidy, a genetic catastrophe that results in spontaneous abortion or birth defects if it arises in the gametes and that is a major contributor to gene dosage imbalances in almost all human cancers. The centromere is the locus on each chromosome that directs accurate chromosome segregation at cell division in healthy cells. The focus of this project is to address two related major questions regarding centromeres: what is the nature of the epigenetic mark that specifies human centromeres and how is this epigenetic mark propagated through cell divisions? Centromeres are the chromosomal loci that confer genetic stability at cell division, but the DNA sequences typically found at the loci are neither necessary nor sufficient for centromere function. Rather, centromeres are specified in metazoans and many other eukaryotes through an epigenetic process. Key to epigenetic centromere specification is a histone H3 variant, CENP-A, and in this proposal we now build on the major findings we made in the previous funding cycle regarding its structure/dynamics, cell cycled-coupled chromatin assembly, and the organization and recruitment of centromere components it directs to human centromeres and neocentromeres. We have identified three critical areas of investigation that are now ripe for eliciting major insight at centromeres: 1) identifying the majo form of the histone/DNA particle into which it assembles at functional human centromeres-representing the fundamental unit of centromeric chromatin and the prime candidate to epigenetically specify centromere location, 2) determining the molecular basis for the cell cycle-coupled program of centromere inheritance, and 3) investigating the stability of CENP-A nucleosomes at individual centromeres and the role therein of other centromere constituents. We will pursue these topics using biochemical, genomic, proteomic, molecular genetic and cell biological approaches, and together these studies have the promise to generate valuable insight into the epigenetic mechanisms that specify and maintain centromere location on the chromosome.